Cryptococcus neoformans is a major pathogen in immunocompetant as well as immunocompromised patients including those with AIDS in both the developed as well as the developing world. Our long-term objective is to test the hypothesis that molecular regulators of the virulence factor laccase affect the virulence of Cryptococcus neoformans. The specific hypothesis behind the present proposal is that a virulence associated DEAD-box protein, Vad1, identified by insertional mutagenesis, is an important regulator of laccase and virulence in C. neoformans. In 2011, we completed our work demonstrating a role for Vad1-dependent degradation of the MFalpha mating pheromone and its role in accelerating the time to successful mating. Mating is an example of a time-critical phenotype where the organism in the wild may not have much time to initiate and complete sexual development before physical elements may separate the mating pairs. We described a new principle whereby transcripts are continually synthesized and degraded to repressed levels when not needed, a so-called transcriptional futile cycle. This is similar to previously-described metabolic cycles that were thought to be futile because metabolic intermediates were produced far in excess to that needed and then were simultaneously degraded, with an apparent futile waste in energy. However, by continually maintaining high level production of these intermediates, cells maintain an ability to rapidly mobilize metabolites by combining an increase in synthesis with an inhibition of degradation. In the transcriptional/degradation cycle described in C. neoformans, degradation of mating pheromone was demonstrated to be altered during induction, to produce a rapid increase in transcript levels in this time critical process. This principal is akin to that used by a race car driver who simultaneously revs the engine, while holding the brake. When given a signal, the driver pops the clutch to more rapidly accelerate his car. In the case of mating of Cryptococcus, we showed that mating was delayed, but not prevented, when the transcriptional clutch was not activated, demonstrating a role for the principle in this time-critical attribute. This principal identifies novel targets for anti-fungal targets in the control of this important human disease. We have also identified additional pathways critical to the regulation of virulence of C. neoformans including a new transcription factor involved in the regulation of cell wall integrity as well as laccase. Cell wall integrity is critical to virulence and is a target in a number of successful anti-fungal agents including the echanocandin class of antifungals. In these studies, we identified a transcription factor, Sp1 that shows an evolutionary similarity to higher metazoans such as humans, vs. that of the yeast factor. We showed that, while the yeast factor is regulated by calcineurin, the cryptococcal factor was regulated by protein kinase C under starvation conditions as demonstrated by a dependence on this pathway for phosphorylation and nuclear translocation and is required for virulence of the organism.